Mitochondrial transfer from Adipose stem cells to breast cancer cells drives multi-drug resistance

Background Breast cancer (BC) is a complex disease, showing heterogeneity in the genetic background, molecular subtype, and treatment algorithm. Historically, treatment strategies have been directed towards cancer cells, but these are not the unique components of the tumor bulk, where a key role is played by the tumor microenvironment (TME), whose better understanding could be crucial to obtain better outcomes. Methods We evaluated mitochondrial transfer (MT) by co-culturing Adipose stem cells with different Breast cancer cells (BCCs), through MitoTracker assay, Mitoception, confocal and immunofluorescence analyses. MT inhibitors were used to confirm the MT by Tunneling Nano Tubes (TNTs). MT effect on multi-drug resistance (MDR) was assessed using Doxorubicin assay and ABC transporter evaluation. In addition, ATP production was measured by Oxygen Consumption rates (OCR) and Immunoblot analysis. Results We found that MT occurs via Tunneling Nano Tubes (TNTs) and can be blocked by actin polymerization inhibitors. Furthermore, in hybrid co-cultures between ASCs and patient-derived organoids we found a massive MT. Breast Cancer cells (BCCs) with ASCs derived mitochondria (ADM) showed a reduced HIF-1α expression in hypoxic conditions, with an increased ATP production driving ABC transporters-mediated multi-drug resistance (MDR), linked to oxidative phosphorylation metabolism rewiring. Conclusions We provide a proof-of-concept of the occurrence of Mitochondrial Transfer (MT) from Adipose Stem Cells (ASCs) to BC models. Blocking MT from ASCs to BCCs could be a new effective therapeutic strategy for BC treatment. Supplementary Information The online version contains supplementary material available at 10.1186/s13046-024-03087-8.


Background
Breast cancer (BC) is the second most prevalent cancer in the female population worldwide.The treatment algorithm of both early and advanced BC still includes chemotherapy in many therapeutic settings [1,2].However, many patients do not respond due to primary or acquired resistance [3].BC cells (BCCs) are surrounded by mammary adipose tissue and intermingled with a repertoire of stromal cells such as adipose stem cells (ASCs), mesenchymal stem cells (MSCs), cancer-associated fibroblasts (CAFs) with endothelial and immune cells, constituting BC microenvironment (BCME), deeply influencing disease development, progression and treatment response [4].Interestingly, the adipose component is altered in BC patients, due to strong immune cells infiltration and chronic inflammatory status [5].MSCs/ASCs play a dominant role in reshaping BCME, promoting epithelial-to-mesenchymal transition (EMT) and supporting cancer stem cells (CSCs), which are, in turn, associated with multi-drug resistance (MDR) [6].The role of ASCs in MDR was highlighted in a recent study where breast adipose tissue-derived ASCs showed a higher potential to enrich CSCs in BC, that, in turn, led to drug resistance [7].
A recent study showed that intercellular mitochondrial transfer (MT) in adipose tissue represents a mechanism of cellular communication regulating systemic metabolic homeostasis [10].Also, MT was reported to influence multiple myeloma features in terms of metabolic switch, reactive oxygen species (ROS) homeostasis and drug sensitivity [11].
One of the major mechanisms responsible for MDR involves adenosine-5'-triphosphates (ATP) binding cassette (ABC) transporters, that can efflux several drugs outside the cell, decreasing intracellular drug concentrations and rendering treatment ineffective.The main efflux transporters involved in BC MDR are ABCB1 (also termed P-glycoprotein, P-gp, or MDR1), multidrug resistance protein 1 (MRP1/ABCC1) and ABCG2 (also termed breast cancer resistance protein BCRP or mitoxantrone resistance protein MXR) [12,13].As these transporters are energy-consuming pumps, they demand considerable amounts of ATP.Remarkably, respirationdeficient BCCs can uptake healthy mitochondria from stromal cells, restoring their functional respiration [14].
Of note, a recent study showed that mitochondrial ATP is the main source of energy for drug efflux in chemo-resistant cancer cells [15].Interestingly, treatment-resistant CSCs are known to be more dependent on oxidative phosphorylation (OXPHOS) and hence have more functional mitochondria and ATP content than their differentiated progeny [16].
These evidences demonstrate a fundamental role of metabolic rewiring in BC, strongly influenced by tumor microenvironment, where stromal cells regulate the consumption and secretion of metabolites, thus modulating the intrinsic metabolic profile of cancer cells [17].
Here, employing cell lines and patient-derived cultures from BC patients, we showed that ASCs transfer their mitochondria to BC cells via MT.Furthermore, we characterized the functional effect of MT, showing that it induces metabolic rewiring and increased ATP production that fuels ABC transporters efflux activity, thereby driving MDR.As a proof-of-concept, we showed that blocking MT restores drug sensitivity, highlighting it as a new potential target for drug development in BC.

Cells and cell cultures
MCF-7, MDA-MB.231 and hASC52telo hTERT cells were purchased from ATCC.The naturally immortalized BC cell line BCAHC-1 was donated by the Pharmacology Department of the University of Calabria [18].MCF-7 and MDA-MB.231cells were cultured in DMEM with glutamine, penicillin, streptomycin, and fetal bovine serum (FBS).BCAHC-1 cells were cultured in DMEM/F-12 with FBS.hASC hTERT were cultured in Mesenchymal Stem Cell Basal Medium for Adiposederived MSCs (ATCC) with Mesenchymal Stem Cell Growth Kit for Adipose-derived MSCs (ATCC, Manassas, Virginia).For hypoxia condition, where expected, we treated the cells with Cobalt Chloride (CoCl 2 ) 100uM (Merck, Milan Italy).All cell lines were kept at 37 °C in a humidified atmosphere with 5% CO2 under mycoplasma-free conditions (checked every three months).

Adipose stem cells isolation
pdASCs were isolated from subcutaneous adipose tissue from abdomen or breast of consenting healthy female patients (mean age = 37 ± 2.5 years).Tissue was digested with collagenase type I/dispase and cells were phenotypically validated according to our previous article [19].The cells were incubated at 37 °C under 5% CO 2 and the medium changed twice a week.

Generation of patient-derived organoids
The BCC-66 patient-derived organoids (PDOs) were generated from a consenting BC patient following a previously published protocol [20].Fresh tissue sample, collected in November 2023, underwent enzymatic digestion with Liberase TL and Y-27632.The obtained cells were embedded in basement membrane matrix (BME) and supplemented with complete medium (Supplementary Table 1).
To generate a 2D/3D hybrid co-culture model, PDOs were trypsinized and plated without BME.After 48 h, having reached a similar volume they were pre-labelled with CellTracker ™ Blue Dye and plated on the top of MitoTracker ™ Green/Red ASCs, at the same conditions described before.
MT evaluation in the 2D/3D co-culture was performed via confocal microscopy.BCC-66 were plated on slide coverslips and co-cultured as previously described.After incubation, samples were fixed and stained with Phalloidin-FITC.Images were captured on a Carl Zeiss LSM 700 Confocal Laser Scanning Microscope (Zeiss LSM 700, Oberkochen, Germany) through a 63x/1.4Pla-nApo immersion objective and analysed for the Z-stach orthogonal view reconstruction with ImageJ software.
For MCP qualitative evaluation, CellTracker ™ Blue Dye MCF-7 and MDA-MB.231were plated on slide coverslips, subjected to MCP, and imaged via confocal microscopy, followed by co-localization statistical analysis.
For ABC transporter evaluation, cells were treated with Doxorubicin (DOX) (Merck, Milan Italy) (1 µM for MDA-MB.231; 2 µM for MCF-7) for 6 h at 37 °C.P-Glycoprotein, ABCG2, and ABCC1 Polyclonal Antibody (all from Elabscience, Houston, USA) were added after blocking and incubated overnight at 4 °C.Cells were washed and incubated with Chk pAB to Rb IgG FITC (Abcam) and stained with Hoechst 33,342, Trihydrochloride, Trihydrate.Samples were imaged on EVOS M5000 Cell Imaging System.All the microscopy assays have been performed three times in triplicate.

Digital holographic microscopy
For kinetic dose-response assay, a Holomonitor M4 microscope (Phase Holographic Imaging AB, Lund, Sweden) was used.Cells were seeded at a density of 10 4 cells/well and then subjected to MCP before treatment with DOX (1 µM for MDA-MB.231; 2 µM for MCF-7).Cells were continuously monitored for 24 h, in time-lapse mode (every hour), at multiple positions in each well using a high-precision motorized stage.The Holomonitor App suite cell imaging software was used for image analysis, for the evaluation of the cell viability parameters (cell density = #cells/cm 2 ; percentage of confluence = %confluence).The results are showed as changes relative to zero time-point, from each area and presented as mean ± SE for all the monitored areas in each well.The assay has been performed three times in triplicate.

Extracellular flux analysis
Oxygen consumption rate (OCR) was analyzed under basal conditions and in response to sequential injections of oligomycin (2 µM), FCCP (2 µM) and rotenone with antimycin A (0.1 µM each) using the Mitochondrial Respiration XF Cell Mito Stress Test.Assays were performed using manufacturer recommended medium (DMEM, 10 mM glucose, 2 mM glutamine, and 1 mM pyruvate, pH 7.4).OCR evaluations were performed with Agilent Seahorse XF Analyzers 24 h after MCP and 2 h after hypoxia.Analysis assay was performed with the Agilent Seahorse Wave Pro software and data analyzed with the Agilent Seahorse Analytics from the Agilent Seahorse XF Analyzer.The assay has been performed three times in triplicate.

Statistical analysis
Cell viability and FACS data were analyzed with Ordinary Two-Way Anova model, adjusted with Bartlett's test, with Tukey's multiple comparison test.MCP analysis was performed with Li's statistical model, based on the evaluation of the intensity correlation quotient (ICQ) [22].This value was obtained by counting the positive voxels, normalized on the total number of voxels (related to cell confluence).Five view fields were analyzed for each sample.Statistical significance for Holomonitor data was determined by the Ordinary Two-Way Anova model, adjusted with the Sidak multiple comparison test.Unless otherwise specified statistical analysis were performed with GraphPad Prism9.Two-tailed p values < 0.05 were considered significant.
Cyt-B very efficiently inhibited TNTs scaffolding (Fig. 1h-k), with dramatic effects on cell morphology.Indeed, cells' structural collapsing was detected, with  1b is the merge of two fields of view, required for the capture of the entire TNTs length).c-d Flow cytometry analysis of the cell fluorescence, for the quantization of the MT occurring from the pre-stained MitoTracker-FITC pdASCs to the pre-labelled CellTracker-Blue MCF7 or MDA-MB.231.The co-culture has been set up in presence or not of a multi-well insert that avoided the cell-to-cell contact (****P ≤ 0.0001).e-f Flow cytometry analysis of the pdASCs mitochondria fluorescence, into the recipient BCCs subset, after treatment with Antimycin A (100 nM), Cyt-B (2,5 uM), CCCP (5 uM) and MdiVi-1 (10 uM).g MTT assay for the evaluation of the viability of the cells used, in our co-culture system, after treatment with Cyt-B.For all the cell lines, the viability rate at 2,5 uM was higher than 85%.h-i Fluorescence microscopy of the co-culture with CellTracker-Blue pre-labelled MCF7 or j-k MDA-MB.231,for the detection of the F-Actin (FITC) and β-Tubulin (TRITC) in presence of Cyt-B (2,5 uM).The yellow arrows point-out the TNTs structures between the two kinds of cells (h-i) or the cytoplasmic F-actin aggregates (j-k) in both BCCs and pdASCs.(****P ≤ 0.0001) smaller appearance, more rounded shape, many membrane invaginations and accumulation of F-actin aggregates in the cytoplasm (Fig. 1j-k).Consequently, cells unable to scaffold the actin-based TNTs could not contact each other, and this impeded MT.
Furthermore, we studied MT in BCC-66 PDOs isolated from a luminal breast cancer patient.Preliminarily, we showed that BCC-66 shared the same genomic profile of the original patient tissue (Supplementary Fig. 2c).According to our previous results, we built a hybrid 2D/3D co-culture between BCC-66, freed from BME, and pdASCs.BCC-66 were able to contact pdASCs, and massively acquired mitochondria from them (Fig. 2g, yellow arrows), although we did not capture TNTs formation.3D orthogonal Z-stack image reconstruction confirmed that the adherent pdASCs directly contact BCC-66 in suspension (Fig. 2g, white arrow).MT was confirmed by FACS, but only in case of physical contact between the cells (****P ≤ 0.0001).Conversely, Cyt-B 2 µM did not significantly inhibit MT (Fig. 2h; Supplementary Fig. 2d), indicating that, in this hybrid 2D/3D co-culture, other mechanisms depending on physical contact could mediate MT.

MitoCeption allows to increase mitochondria acquisition
To better distinguish the selective effect of exogenous mitochondria transferred to BCCs, we employed the MitoCeption (MCP), a procedure used to force mitochondria internalization, derived from a donor into a recipient cell that functionally engulf and retain them in its cytoplasm.According to Caicedo et al. [21], we isolated and characterized mitochondria from both pdASCs and hTERT immortalized hASCs; then we transferred them to MCF-7 or MDA-MB.231cells (Fig. 3a).After 24 h, MCP was validated by Z-stack orthogonal view reconstruction of confocal images.Here, we identified pdASC pre-labelled mitochondria (MitoTrackerred) spatially distributed in the cytoplasm of the BCCs (CellTracker-DAPI) (Fig. 3b).MCP efficiency was evaluated verifying mitochondria internalization by statistical analysis of the co-localization between the transferred pdASCs mitochondria and the recipient cells (Fig. 3c).After 24 h (T 24h ) mitochondria co-localized with cells, as shown by analysis of the not-random spatial correlation.Moreover, MDA-MB.231 were significantly more able to intake exogenous mitochondria than MCF-7 (****P ≤ 0.0001) (Fig. 3c).
Taken together, our results show that MCP allows to increase the efficiency of mitochondria uptake and that MDA-MB.231acquire significantly more mitochondria than MCF-7.

ASCs-derived mitochondria potentiate BC multi-drug resistance
To functionally characterize MT effect, we investigated the antitumoral activity of different chemotherapy (See figure on next page.)Fig. 2 MT occurs both between pdASCs and human primary 2D and 3D cell models.a Fluorescence microscopy (magnification 100X) of pre-labelled BCAHC-1 (CellTracker-Blue) and pre-stained pdASCs (MitoTracker-Red) with F-Actin (Phalloidin-FITC).Yellow arrows point-out the pdASC mitochondria along the TNTs and into recipient BCAHC-1 cell.b Flow cytometry analysis of the cell fluorescence, for the quantization of the MT occurring from the pre-stained MitoTracker-FITC pdASCs to the pre-labelled CellTracker-Blue BCAHC-1.The co-culture has been set up in presence or not of a multi-well insert that avoided the cell-to-cell contact (****P ≤ 0.0001).c MT inhibition in co-culture between pdASCs and BCAHC-1.Flow cytometry analysis of the pdASCs mitochondria fluorescence, into the recipient BCAHC-1 subset, after treatment with Antimycin A (100 nM), Cyt-B (2,5 uM), CCCP (5 uM) and MdiVi-1 (10 uM) d MTT assay for the evaluation of the BCAHC-1 viability after treatment with Cyt-B.e-f Fluorescence microscopy of the co-culture with CellTracker-Blue pre-labelled BCAHC-1, for the detection of the F-Actin (FITC) and β-Tubulin (TRITC) in presence of Cyt-B (2,5 uM).The yellow arrows point-out the TNTs structures between the two kinds of cells (e) or the cytoplasmic F-actin aggregates (f) in both BCAHC-1 and pdASCs.(****P ≤ 0.0001).g Confocal microscopy 3D orthogonal reconstruction, z-stack technology of a 2D/3D hybrid coculture shows the pre-labelled BCC-66 (CellTracker-Blue) and pre-stained pdASCs (MitoTracker-Red) with F-Actin (Phalloidin-FITC).The white arrows in the YZ plane point-out that the pdASCs are in contact with the BCC-66 and the yellow arrow indicates the presence of exogenous mitochondria in the cytoplasm of BCC-66.h Flow cytometry analysis of the cell fluorescence, for the quantization of the MT occurring from the pre-stained MitoTracker-FITC pdASCs to the pre-labelled CellTracker-Blue BCC-66, also after treatment with Cyt-B (2 uM).The co-culture has been set up in presence or not of a multi-well insert that avoided the cell-to-cell contact agents on BCCs carrying exogenous mitochondria.Considering that the MCP significantly downregulated HIF-1α protein expression in the recipient BCCs (Fig. 4a; Supplementary Fig. 3a-b), we decided to adopt this condition in our experiments.
Here, the DTX strongly promoted apoptosis, even in hypoxia, with a significant ADM-mediated reversion of the phenomenon.
Taken together, our results indicate that ADM increased MDR in BCCs preventing the induction of apoptosis, in both hypoxic and normoxic conditions.

ASCs-derived mitochondria induce an increase of ATP production in BCCs
Since mitochondria play a pivotal role in energy homeostasis, we evaluated the effect of ADM on BCCs metabolism, via extracellular flux analysis.Here, ADM strongly upregulated ATP rate production, associated with mitochondrial respiration (Δ ATP ).Indeed, in ADM-carrying MCF-7 we found a significantly higher basal (OCR > 18%) and maximal respiration (OCR > 10%), with a relative increase of ATP production rate (Δ ATP > 35 pmol/min; ****P ≤ 0.0001) (Fig. 4f ).Furthermore, we found a significant reduction of the spare respiration (OCR < 18%), mainly due to the increased basal oxygen consumption rate mediated by ADM.On the contrary, in hypoxia ADM did not significantly influence basal nor maximal respiration, however the total oxygen consumption capacity was raised, leading to a relevant increase of ATP production (Δ ATP > 27 pmol/min; ****P ≤ 0.0001) (Fig. 4g).In MDA-MB.231 the effect of ADM on mitochondrial metabolism was very similar.In these cells, the basal respiration was significantly increased after MCP (OCR > 12%), without any significant difference on the maximal respiration (Fig. 4h).Even in this case, ADM significantly reduced the spare respiration (OCR < 18%), due to basal oxygen consumption rate increasing, which led to the doubling of ATP production rate (Δ ATP > 90 pmol/ min; ****P ≤ 0.0001).In hypoxic MDA-MB.231 the respiratory capacity as well as the basal respiration didn't significantly change after MCP (Fig. 4i).Nevertheless, both these parameters increased (OCR > 12%) contributing to significantly enhancing the ATP production rate (Δ ATP > 47 pmol/min; ****P ≤ 0.0001).Taken together, our results indicate that ADM led to an increase in ATP production.

ASCs-derived mitochondria activate the ABC transporter-mediated drug efflux
As ATP-binding cassette (ABC) transporters are involved in BC MDR, we evaluated their role in our MCP model, focusing on P-gp, ABCG2 and ABCC1 [13] by fluorescence microscopy and FACS 8 h after the induction of hypoxia, and subsequent treatment with DOX.We chose DOX both to take advantage of its autofluorescence and because it is a backbone of chemotherapy treatment in BC [15].
In MCF-7, we observed that P-gp and ABCC1 expression increased after DOX treatment in both oxygen conditions, while ABCG2 increased after DOX only in normoxia (Fig. 5a-c; Supplementary Fig. 5).In MDA-MB.231P-gp expression was increased after DOX (in both oxygen conditions) and in hypoxia only.ABCG2 was not influenced by hypoxia and increased only with DOX treatment in both oxygen conditions while ABCC1 only increased in normoxia after DOX treatment (Fig. 5d-f; Supplementary Fig. 5).
Subsequently, we focused our attention on the possible effect of ADM on ABC transporters activity.Preliminarily, we evaluated DOX cytotoxicity on BCCs after MCP.Indeed, it induced an increase of cell viability parameters monitored with Holomonitor (***P ≤ 0.001), also in hypoxia (**P ≤ 0.01) (Supplementary Fig. 6a-b).In MDA-MB.231,we achieved the same results in normoxia (#cells/cm 2 = ***P ≤ 0.001; %confluence = **P ≤ 0.01) and hypoxia (#cells/cm 2 = **P ≤ 0.01; %confluence = *P ≤ 0.05) (See figure on next page.)Fig. 4 MCP increases BCCs viability under chemotherapy treatment and impacts on their metabolism, modifying mitochondrial respiration.a HIF-1α expression analysis in BCCs subjected to MCP, in N-OX and H-OX conditions.The MCP significantly reverted the up-regulation of HIF-1α in the BCCs after their stimulation with the chemical hypoxia inducer cobalt chloride (100 uM).b-c Cell viability and apoptosis assay d-e of the BCCs subjected to MCP in different oxygen conditions, after treatment with the chemotherapeutic drugs DTX (50 nM) or CIS (10 uM) (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; ****P ≤ 0.0001).f-g SeaHorse ATP rate assay for the analysis of the mitochondrial respiration in MCF-7 and (h-i) MDA-MB.231,24 hours after the MCP, in different oxygen conditions.The histograms represent the ATP production rate (pmol/min), calculated during the oxygen consumption phase (****P ≤ 0.0001) (Fig. 6a-d).Thereafter, we evaluated DOX cytoplasmic retention by measuring the DOX MFI .As expected, the ABC-transporter inhibitor VER increased DOX cytoplasmic accumulation [23].Nevertheless, ATP metabolism was crucial in regulating the activity of these transporters.Indeed, in both oxygen conditions, the ATP synthesis inhibitor Rotenone strongly reduced ABC transporters efflux capacity, and this effect was significantly reverted by D-Glu (Fig. 6e-h; Supplementary Fig. 7).These results highlight that ABC transporter activity is strictly dependent on ATP availability, independently from cell respiration.
Taking all these results into consideration, we showed that ADM activated ABC transporters into both MCF7 and MDA-MB.231cell lines, leading to a significant reduction of drug accumulation within the cytoplasm and escape from chemotherapy induced cytotoxicity.

Discussion
Recent studies demonstrated that BC development and progression as well as treatment response depends also on its complex micro-environment [24,25].MSCs are recruited to the site of tumor formation, where they promote a more aggressive phenotype, in terms of acquired/ enriched stemness, chemoresistance [26] and distant dissemination [5].ASCs influence BCCs through the secretion of cytokines, chemokines and/or growth factors involved in cell proliferation and migration, inflammation and angiogenesis [27].Moreover, ASCs also interact with BCCs either activating an intense vesicular trafficking [28] or directly by "cell-cell" connections.In this spatial configuration, ASCs can interact with BCCs through different mechanisms, such as receptor/ligand interactions [29], cell-cell fusion processes [30,31] or TNTs formation [32,33].Indeed, in a previous study, we showed that BCCs are able to maintain hASCs in a 'stem state' in vitro, while hASCs promote tumor angiogenesis in vivo, favoring tumor growth and aggressiveness [19].
Mitochondria are cytoplasmic organelles that provide numerous bioenergetic and biosynthetic processes, whose dynamics and activity are strictly regulated.They play a pivotal role in the maintenance of cancer cell homeostasis, during tumor progression, because of their ability to favor the adaptive cell response to adverse conditions, such as oxidative stress, hypoxic environment or chemotherapy induced cytotoxicity [34].In cancer cells mitochondria switch to a more fused pattern [35,36], can promote the activation of many anti-apoptotic pathways, also favoring a higher ATP production linked to the respiratory chain, thanks to an increased mitochondrial cristae density [37].
In this study, we demonstrated the occurrence of mitochondrial transfer from ASCs to BCCs, that promotes an adaptive metabolic response and chemoresistance in the recipient cells.We developed different 2D cocultures, employing commercial cell lines belonging to different immunophenotypes, pdASCs and also patient-derived breast cancer models.We highlighted the formation of a complex TNTs network between pdASCs and adherent BCCs (MCF-7, MDA-MB.231 and the primary BCAHC-1), showing that this occurs in both luminal and triple negative models, as well as in patient-derived models.These membrane projections exhibit highly variable morphology, in terms of thickness and length, and are full of mitochondria.Connection via TNTs allows the trafficking of macro-molecules and/or of entire organelles, in particular mitochondria, between MSCs and cancer cells [38][39][40].Here we showed that this TNTs allows to transfer mitochondria from pdASCs to BCCs (MCF-7 and MDA-MB.231).In these models we showed that MT was dependent on TNT's formation, as highlighted by its abolishing when disrupting actin polymerization.These achievements were confirmed also with the adherent patient-derived primary BC cell line BCAHC-1.Indeed, even in this co-culture model, primary BCCs acquired mitochondria from pdASC, in a process strictly related to cytoskeleton remodeling.Furthermore, Cytochalasin B was the only drug that affected MT in all 2D cell lines, while the other gave differential effects.Moreover, MT was confirmed also from pdASCs to BCC-66, a patientderived organoid model developed in our laboratory, showing that the process occurs also in a more physiological context.Nevertheless, in this hybrid 2D/3D coculture, the TNTs scaffolding blockade did not significantly affect MT, demonstrating that in this complex spatial model it occurs through additional cell-cell contactdriven mechanisms that deserve further investigation.
From a functional point of view, we characterized the biological effect mediated by the acquisition of exogenous mitochondria.To better dissect this aspect from the background noise, due to the plethora of cell-cell interactions in co-culture, we decided to adopt the MCP [21].Thanks to this approach, we showed that ADM uptake modifies the cell oxygen balance, completely reverting the hypoxic status.In fact, ADM impressively reduced the expression of HIF-1α in our models subjected to inducible hypoxia.According to these results, we evaluated the relationship between ADM and BC drug resistance, also considering that BCCs immunophenotype significantly influences their sensitivity to the different chemotherapies.Indeed, some authors demonstrated that ERα-positive cells, such as MCF-7 are less sensitive to anthracyclines than ERα-negative cells, such as MDA-MB.231[41].Furthermore, Caicedo et al., showed that the internalization of MSCs-derived mitochondria into BCCs led to an increased OXPHOS and acquisition of drug resistance [21,42], showing that metabolic rewiring can influence drug response.In our study, ADM promoted a metabolic switch in recipient cells.Indeed, it significantly improved MCF-7 basal mitochondrial respiratory activity in normoxic as well hypoxic conditions, increasing the oxygen capacity and the relative OXPHOS.Conversely, MDA-MB.231, that prominently depend on glycolysis, showed a switch to OXPHOS, with a significant increase of ATP production, mainly in normoxic conditions.
Since MCP induced cancer cells to be more resistant to chemotherapy, and the ABC drug efflux transporters are among the main proteins involved in MDR [43], we investigated on a possible cause-effect relationship between the MCP-induced metabolic switch and ABC transporter functionality.We employed docetaxel and anthracyclines which are universally employed drugs for the treatment of breast cancer in all disease settings and subtypes according to international guidelines, and cisplatin as platinum salts are employed for triple negative breast cancer.Recently, a BCCs chemo-resistant sub-population was described, with an elevated mitochondrial respiratory capacity due to the loss of Methylation-Controlled J protein (MCJ), an endogenous negative regulator of mitochondrial activity.The ATP production boost, observed in these models, fueled the ABC transporters which increased their efflux ability [15].In our model, we found high expression levels of P-gp, ABCG2 and ABCC1, the ABC transporter isoforms which are prominently involved in BC-MDR [18,44,45].Some studies correlate P-gp high protein expression with a higher metastatic potential in triple negative BC, while ABCG2 over-expression is significantly linked with a better prognosis [46].Indeed, we found a high basal expression of these transporters, especially in the triple negative MDA-MB.231, but also in luminal MCF7.Moreover, we confirmed that hypoxia and the pressure exerted by chemotherapy positively regulated the expression of these efflux pumps, as previously demonstrated [47].
Thereafter, taking advantage of our purer model developed thanks to MCP, we investigated the impact of MT on ABC transporters functionality.Interestingly, we found that ADM significantly increased the DOXO efflux ability of MCF-7 and MDA-MB.231 in both oxygen conditions, but with a stronger effect in hypoxia, a condition that is more representative of the real tumor oxygen status.
Specifically, we demonstrated that MCP metabolically boost the BC recipient cells, leading to the intracellular increasing of ATP, a direct molecular activator of the ABC transporters.These efflux pumps play a key role in the BCCs MDR, as demonstrated in our models, where we found a strongly increased intracellular DOX retention in both oxygen conditions, by selectively blocking ABC receptors with VER [48].
Our results show that the increased ATP generated after ADM uptake is the key modulator of the ABC receptor efflux in BCCs.Indeed, when we blocked ATP synthesis with rotenone, we saw an increase of DOX cytoplasmic retention, indicating a downregulation of ABC transporters function.On the other side, the administration of D-Glu reversed this inhibition, reactivating ABC efflux activity (Fig. 7).

Conclusions
In conclusion, we demonstrate for the first time that MT promotes extensive BCCs changes leading to a more resistant phenotype.We discovered that pdASCs interact with BCCs, donating their own mitochondria.This MT is strictly related to cell structure remodeling, that allows the physical cell-cell contact.This mechanism drives many functional effects in the recipient cancer cell, that benefit from this process.Indeed, we showed that these cells improved their adaptive response to the extracellular environment, increased their mitochondrial respiration and the relative OXPHOS and upregulated ABC transporter activity, thereby acquiring a phenotype that better escape from anticancer therapies.Intriguingly, we also demonstrated the MT in more accurate and translational in vitro models that reproduce some key aspects of the tissue of origin.Furthermore, we show for the first time that inhibiting MT could effectively restore sensitivity to chemotherapy making it a new potential target to develop innovative treatment strategies.Further studies are needed to better elucidate the occurrence of alternative ways for cancer cells to attract exogenous mitochondria, but this study opens the way to new treatments for breast cancer, either by blocking the MT mechanisms from ASCs to BCCs or by inhibiting the interaction between mesenchymal stem cells and tumor cells.

Fig. 1
Fig. 1 MT occurs between pdASCs and BCCs via TNTs and Actin polymerization inhibition disrupts TNTs inhibiting MT. a-bFluorescence microscopy of pre-labelled MCF7 or MDA-MB.231(CellTracker-Blue) and pre-stained pdASCs (MitoTracker-Red) with F-Actin (Phalloidin-FITC).The yellow arrows point-out the pdASC mitochondria along the TNTs and into recipient BCCs (100X; Fig.1bis the merge of two fields of view, required for the capture of the entire TNTs length).c-d Flow cytometry analysis of the cell fluorescence, for the quantization of the MT occurring from the pre-stained MitoTracker-FITC pdASCs to the pre-labelled CellTracker-Blue MCF7 or MDA-MB.231.The co-culture has been set up in presence or not of a multi-well insert that avoided the cell-to-cell contact (****P ≤ 0.0001).e-f Flow cytometry analysis of the pdASCs mitochondria fluorescence, into the recipient BCCs subset, after treatment with Antimycin A (100 nM), Cyt-B (2,5 uM), CCCP (5 uM) and MdiVi-1 (10 uM).g MTT assay for the evaluation of the viability of the cells used, in our co-culture system, after treatment with Cyt-B.For all the cell lines, the viability rate at 2,5 uM was higher than 85%.h-i Fluorescence microscopy of the co-culture with CellTracker-Blue pre-labelled MCF7 or j-k MDA-MB.231,for the detection of the F-Actin (FITC) and β-Tubulin (TRITC) in presence of Cyt-B (2,5 uM).The yellow arrows point-out the TNTs structures between the two kinds of cells (h-i) or the cytoplasmic F-actin aggregates (j-k) in both BCCs and pdASCs.(****P ≤ 0.0001)

Fig. 3
Fig. 3 Construction and validation of MitoCeption model.a MCP assay workflow, from pdASCs/hASCs hTERT mitochondria-derived isolation to the forced engulfment into the BCCs.b Confocal microscopy 3D orthogonal reconstruction, with z-stack technology, of the pre-labelled CellTracker-Blue BCCs (MCF-7) after the MCP with MitoTracker-FITC pre-labelled pdASCs derived mitochondria (c) Fluorescence microscopy and spatial co-localization statistical analysis of BCCs and MitoTracker stained pdASCs derived mitochondria at time points 0 h and 24 h (Li's ICQ value analysis normalized on the total cells area; ****P ≤ 0.0001)

Fig. 7
Fig. 7 Proposed model of ABC transporter regulatory mechanism after ADM uptake